This research's long-term objective is to understand ion regulation in insects, particularly in mosquitoes. Our present grant has determined the structure of many key proteins involved in ion regulation in Aedes aegypti Malpighian tubules. Further we have preliminary data that show enhanced V-ATPase transcript levels following a blood meal. In the present proposal we plan to build on these findings. Our objective in this proposal in to molecularly characterize the proteins that are involved in Na+ and C1- transport in mosquitoes. In the first objective we plan to functionally characterize a Na+/K+/2C1- cotransporter that was isolated from A. aegypti Malpighian tubules. This cotransporter is significantly divergent from known mammalian and insect cotransporters. Further while the V-ATPase plays a crucial role in ion regulation in mosquitoes, other proteins facilitate actual cation transport. Hence in the second objective we will also characterize the cation/antiporter CDNA clone isolated from the midgut and/or Malpighian tubule of A. aegypti. In the third objective we will attempt to evaluate changes in Malpighian tubules function following a blood meal. Therefore we will follow up our preliminary studies on V-ATPase regulation. We will determine shether V1 and V0 dissociation- reassociation occurs in Malpighian tubules after blood feeding ans also whether these genes are transcriptionally regulated. Data from these experiments will help elucidate mechanisms of change in ion transport in mosquito Malpighian tubules that occurs following a blood meal. In addition we will evaluate whether the A. aegypti Na+/K+/2C1- cotransporter is regulated. In summary, this proposal is directed towards elucidating the molecular processes involved in ion secretion following a blood meal in mosquitoes. Characterization of these processes may aid future work that could focus on the disruption of these regulatory processes in an important vector of human disease.